Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis, most commonly of the erythroid lineage, resulting in a phenotype termed refractory anemia. Patients with MDS who have a heterozygous, interstitial deletion of chromosome 5q (Del (5q)), have a highly reproducible clinical phenotype, though the molecular basis of this phenotype was previously unknown. In the first funding period of this grant, we found that haploinsufficiency for RPS14, one of the genes within the common deleted region for del (5q) MDS, causes a block in erythropoiesis, the dominant clinical phenotype associated with this genetic abnormality, establishing a previously unrecognized link between del (5q) MDS and Diamond Blackfan anemia, a congenital disorder with a similar phenotype that is also caused by genetic inactivation of one allele of genes encoding ribosomal proteins. In the most recent funding period, we found that haploinsufficiency for CSNK1A1 causes elevated ?-catenin levels, an increase in the number and function of hematopoietic stem cells, and sensitivity to the drug lenalidomide. Moreover, we identified recurrent, gain-of-function somatic mutations in CSNK1A1, the first gene within the Del (5q) common deleted region identified with recurrent somatic mutations. In the current proposal, we first aim to determine the mechanisms underlying clonal dominance of cells with CSNK1A1 heterozygous deletion or mutation using both murine models and phosphoproteomics. We next aim to determine how CSNK1A1 haploinsufficiency interacts with haploinsufficiency for other Del (5q) genes using a cellular barcoding system to perform competitive repopulation assays with multiple genotypes in a single mouse model. Finally, we will investigate the biochemistry of lenalidomide-dependent degradation by the CRL4CRBN ubiquitin ligase, using a novel saturation mutagenesis functional screen. These studies will inform the biology and treatment of Del (5q) MDS, the mechanisms underlying heterozygous deletions in cancer more broadly, and the development of novel therapeutics targeting ubiquitin ligase activity.